The proposed study tests the following hypotheses: 1) An important difference between tumor cells and normal cells is in the capacity to conserve ribonucleotide pools under conditions that limit ribonucleotide synthesis. Decreased conservation capacity of tumor cells may be due to differences in regulation of RNA metabolism by ATP and GTP pools. The evidence supporting this hypothesis is that ATP pools decrease rapidly and profoundly when tumor cells, but not normal cells, are incubated in glucose-free medium. If RNA synthesis in tumor cells is inhibited by actinomycin D prior to incubation of cells in glucose-free medium, the decreases in ribonucleoside triphosphate pools are diminished and the ribonucleotide pools are conserved. ATP and GTP pools decrease in growing tumor cells, but not in normal cells, incubated with methotrexate (MTX). If RNA synthesis is inhibited by actinomycin D or doxorubicin prior to the addition of MTX, ATP and GTP pools in tumor cells do not decrease. The proposed study seeks to confirm these preliminary findings, to measure the rates of RNA synthesis and of RNA degradation in glucose-deprived and in MTX-treated tumor cells and normal cells, and to measure RNA synthesis rates in isolated nuclei of tumor cells and of normal cells as a function of ATP and GTP concentrations. 2) The differential toxicity of MTX and of other chemotherapeutic agents for tumor cells may be due in part to induction of purine-depleted states. ATP and GTP concentrations will be determined in a variety of MTX-treated tumor cells and normal cells. 3) The mechanisms of ATP and GTP depletion in MTX-treated tumor cells are consumption of ATP and GTP pools for nucleic acid synthesis and inhibition of ATP regeneration by glycolysis and mitochondrial oxidative phosphorylation. Detailed studies delineating the relative importance of these processes will be performed as outlined. 4) Purine depletion may be as important as or more important than thymidylate depletion in MTX toxicity for tumor cells. Multiplication capacity of tumor cells will be determined after incubation with MTX in media containing thymidine or purines. 5) ATP-depleted cells may die because of impaired functioning of cell membrane (Na(I) plus K(I)) ATPase, K(I) efflux and influx of Na(I) and water.